There currently exists an unfulfilled need for the production of many specialty chemicals, and liquid fuels, from renewable resources. Despite a great negative impact on the environment, the earth's oceans and atmosphere, the great majority of chemical compounds and motor vehicle fuels are still derived from fossil fuels such as crude oil, coal tar, shale oil and so on. Of particular note, simple phenolic compounds such as the cresols, are largely extracted from coal tar in a process that has such a negative impact on the environment that U.S. factories specializing in cresol production have been closed in recent years. This, however, is not a global solution, as the problem may simply be transferred to another geographical location. Synthetic biology offers potential solutions to the manufacture of certain specialty chemicals such as these.
Polyketides generally are synthesized by condensation of two-carbon units in a manner analogous to fatty acid synthesis. In general, the synthesis involves a starter unit and extender units; these “two-carbon” units are derived from, for example, acylthioesters, typically acetyl, propionyl, malonyl or methylmalonyl coenzyme-A thioesters. There are four classes of polyketide synthases (PKSs) that differ in the “manner” in which the catalytic sites are used (Watanabe and Ebizuka, 2004). The type I modular PKSs are single proteins with multiple “modules” that contain catalytic sites that are used only once in linear “assembly-line” style. The type I iterative PKSs are single proteins with sites that are used repeatedly to reach the final polyketide product. The present invention can employ coding sequences from each of the classes, but in preferred embodiments, the PKSs employed will be derived primarily from this class of PKS, e.g. the aromatic 6-methylsalicylic acid synthases, or the orsellinic acid synthases, or modified versions of these enzymes. Genes encoding polypeptide components of type I PKSs have been used for the microbiological production of polyketides in heterologous microorganisms such as yeast and E. coli. See for example U.S. Pat. Nos. 6,033,883, 6,258,566, and 7,078,233.
The type II PKSs contain multiple proteins, each with a single monofunctional active site. The active sites may be used only once, or repeatedly. Lastly, type III PKSs are single proteins with multiple modules, in which the active sites are used repeatedly.
The PKSs operate in an analogous way to the fatty acid synthases (FASs). Fatty acids are generally composed of hydrophobic components and can also be used as an energy source, such as in biofuels. Many eukaryotes synthesize fatty acids using common or similar metabolic pathways. In seeds, fatty acids, as part of triglycerides, are stored as a source of energy for further germination. The FAS pathway is located in the plastids. Acetyl-ACP (acyl carrier protein) is formed by a condensing enzyme, β-ketoacyl-ACP synthase (KAS) III. Elongation of the acetyl-ACP to longer chain fatty acids involves the cyclical action of the condensation of a 2-carbon unit from malonyl-ACP to form a longer β-ketoacyl-ACP (β-ketoacyl-ACP synthase), reduction of the keto function to an alcohol (β-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (β-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). β-ketoacyl-ACP synthase I (KAS I), is primarily responsible for elongation up to palmitoyl-ACP (C16:0), whereas β-ketoacyl-ACP synthase II (KAS II) is predominantly responsible for the final elongation to stearoyl ACP (C18:0).
Similarly, iterative aromatic PKSs utilize the condensation of a 2-carbon unit from malonyl-ACP to form products such as 6-methylsalicylic acid (6-MSA), also known as 2-hydroxy-6-methylbenzoic acid (HMBA), and orsellinic acid (OSA) via the biosynthetic pathways shown in FIGS. 1A-1B and 2A-2C.
The flexibility of these pathways is underscored by the fact that a bacterial 6-MSAS can be engineered to synthesize OSA simply by “knocking out” the catalytic activity of the ketoreductase (KR) domain (Ding et al., Chemistry & Biology 17, 495-503, 2010).
The present invention provides systems for production of molecules and precursor molecules with multiple uses in the microbicidal, pharmaceutical and renewable liquid fuel areas. In some embodiments, addition of the machinery for pantetheinylation of the acyl carrier proteins (i.e., using a holo ACP synthase, also known as a PPTase) permits production of said molecules in a wide spectrum of hosts that may not necessarily produce such molecules naturally.
The aromatic molecules thus obtained directly or indirectly from the microbial hosts, such as cresols, orcinols, hydroxymethylbenzoic acids and their cognate ethers, esters and lactones, yield homogeneous or heterogeneous preparations of compounds that are, or can be further treated to yield compounds suitably used as microbicidals, pharmaceuticals, vitamins, flavoring agents or renewable energy sources, such as a fuel, a fuel additive, such as an oxygenate, a fuel adjunct, i.e. a high octane gasoline blending agent and so on.